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      Recent technical advances in proteomics

      review-article
      a , 1
      F1000Research
      F1000 Research Limited
      proteomics, mass spectrometry, spatial proteomics, interaction proteomics

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          Abstract

          Mass spectrometry is one of the key technologies of proteomics, and over the last decade important technical advances in mass spectrometry have driven an increased capability for proteomic discovery. In addition, new methods to capture important biological information have been developed to take advantage of improving proteomic tools.

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          Most cited references54

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          Molecular Structure of the Human CFTR Ion Channel.

          The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR's channel function.
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            The Orbitrap: a new mass spectrometer.

            Research areas such as proteomics and metabolomics are driving the demand for mass spectrometers that have high performance but modest power requirements, size, and cost. This paper describes such an instrument, the Orbitrap, based on a new type of mass analyzer invented by Makarov. The Orbitrap operates by radially trapping ions about a central spindle electrode. An outer barrel-like electrode is coaxial with the inner spindlelike electrode and mass/charge values are measured from the frequency of harmonic ion oscillations, along the axis of the electric field, undergone by the orbitally trapped ions. This axial frequency is independent of the energy and spatial spread of the ions. Ion frequencies are measured non-destructively by acquisition of time-domain image current transients, with subsequent fast Fourier transforms (FFTs) being used to obtain the mass spectra. In addition to describing the Orbitrap mass analyzer, this paper also describes a complete Orbitrap-based mass spectrometer, equipped with an electrospray ionization source (ESI). Ions are transferred from the ESI source through three stages of differential pumping using RF guide quadrupoles. The third quadrupole, pressurized to less than 10(-3) Torr with collision gas, acts as an ion accumulator; ion/neutral collisions slow the ions and cause them to pool in an axial potential well at the end of the quadrupole. Ion bunches are injected from this pool into the Orbitrap analyzer for mass analysis. The ion injection process is described in a simplified way, including a description of electrodynamic squeezing, field compensation for the effects of the ion injection slit, and criteria for orbital stability. Features of the Orbitrap at its present stage of development include high mass resolution (up to 150,000), large space charge capacity, high mass accuracy (2-5 ppm), a mass/charge range of at least 6000, and dynamic range greater than 10(3). Applications based on electrospray ionization are described, including characterization of transition-metal complexes, oligosaccharides, peptides, and proteins. Use is also made of the high-resolution capabilities of the Orbitrap to confirm the presence of metaclusters of serine octamers in ESI mass spectra and to perform H/D exchange experiments on these ions in the storage quadrupole. Copyright 2005 John Wiley & Sons, Ltd.
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              Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation.

              Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts. Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation. Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved, and has important consequences for complex formation and regulation of protein abundance.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Writing – Original Draft PreparationRole: Writing – Review & Editing
                Journal
                F1000Res
                F1000Res
                F1000Research
                F1000Research
                F1000 Research Limited (London, UK )
                2046-1402
                29 March 2019
                2019
                : 8
                : F1000 Faculty Rev-351
                Affiliations
                [1 ]Molecular Medicine and Neurobiology, Scripps Research, 0550 North Torrey Pines Road, SR302, La Jolla, CA, 92037, USA
                Author notes

                No competing interests were disclosed.

                Author information
                https://orcid.org/0000-0001-5267-1672
                Article
                10.12688/f1000research.16987.1
                6441878
                30997033
                8c8f0b06-0662-49ac-9b3c-0088aca110ff
                Copyright: © 2019 Yates, III JR

                This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 20 March 2019
                Funding
                Funded by: National Institute of Health
                Award ID: R01MH067880
                Award ID: P41GM133533
                This work was supported by the National Institutes of Health (grants R01 MH067880 and P41 GM133533).
                The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                proteomics,mass spectrometry,spatial proteomics,interaction proteomics

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